10-Formyltetrahydrofolate dehydrogenase (10-FTHFDH; EC 1.5.1.6) catalyzes the NADP-dependent oxidation of formate and excess one-carbon (1-C) units, in the form of 10-formyltetrahydrofolate (10-HCO-H4PteGlu), to CO2 and tetrahydrofolate (H4PteGlu) in liver cytosol. A similar reaction is known to occur in liver mitochondria, but the enzyme which catalyzes the reaction has not been identified. Evidence is presented which indicates that the mitochondrial enzyme is not an isozyme of cytosolic 10-FTHFDH, but is a different enzyme. The mouse strain NEUT2 lacks 1O-FTHFDH and cannot oxidize the ring-C-2 of histidine to CO2. NEUT2 mice are, however, able to oxidize formate at 50% of the rate of normal mice. These observations indicate that there is another, as yet unidentified pathway for formate oxidation. NEUT2 mice have allowed us to demonstrate that an alternate pathway for formate oxidation exists. These mice also offer a unique opportunity to study the effects of a key enzyme deletion on folate nutrition, and folate and 1-C metabolism. The long term objective of this project is to investigate the regulation of folate-mediated disposal of 1-C units, from formate and amino acid catabolism, to CO2 by cytosolic 10-FTHFDH and the as yet unidentified mitochondrial enzyme. The investigation will determine the differences between NEUT2 and normal mice, fully characterize the mitochondrial enzyme and examine formate oxidation in liver. Future investigations will determine if human liver also has the mitochondrial enzyme and if it is involved in formate oxidation. A lack of the mitochondrial enzyme or its inactivity might further explain the susceptibility of humans and primates to methanol and formate toxicity. The specific aims are: 1. The characterization of normal and NEUT2 mice in terms of hepatic folate pools, folate-dependent enzyme activities, purine biosynthesis and the oxidation of formate and other 1-C donating compounds. 2. Purification, characterization and cloning of the mitochondrial enzyme that catalyzes the oxidation of 10-HCO-H4PteGlu to CO2 and H4PteGlu. 3. An investigation of the alternate formate oxidation pathway to determine whether it utilizes the mitochondrial enzyme that catalyzes the oxidation of 10-HCO-H4PteGlu.